The long-term objective of the studies in this proposal is to understand how eukaryotic cells coordinate the large number of proteins involved in replication, repair, and homologous recombination to faithfully duplicate their genome. This complicated process is essential to the maintenance of genome stability, loss of which can lead to cancer or premature aging. Recent studies suggest that a family of proteins, the RecQ-type helicases, might play a role in facilitating DNA replication. In human cells, there are five RecQ family members, three of which are deficient, respectively, in Werner syndrome (WRN), Bloom syndrome (BLM), or a subset of Rothmond-Thompson syndrome (RecQ4). In this proposal, a biochemical approach will be taken to study the roles of WRN, RecQ4, and BLM in replication and, more broadly, the factors and mechanism involved in replication fork restart. The model system to be used is the nuclearplasmic extracts (NPE) derived from nuclei reconstituted in Xenopus egg extracts. This in vitro system recapitulates faithfully the mechanics and regulation of eukaryotic cellular DNA replication. Three specific aims are proposed. The first specific aim seeks to study the roles of FFA-1 (Xenopus WRN) and xRecQ4 (Xenopus RecQ4) in the replication of various defined DNA substrates. The second specific aim seeks to study the role of xBLM (the Xenopus Bloom syndrome protein) in replication fork assembly and characterize the role of topoisomerase 3a (xTopo 3alpha), which interacts with xBLM, in replication. The third specific aim seeks to systematically analyze the factors and mechanism involved in the restart of stalled replication forks using a biochemical system developed in the lab. A variety of methods, including immunodepletion, immunofluorescence staining, affinity protein purification, recombinant protein expression, and tnd biochemical fractionation will be used to accomplish the proposed studies. The results from these studies are expected to significantly advance the understanding of eukaryotic DNA replication fork dynamics and how defective RecQ helicases lead to human diseases like cancer and premature aging.